Treatment of microbial infections

ABSTRACT

The present invention is directed to improved microbial antigen vaccines, pharmaceutical compositions, immunogenic compositions and antibodies and their use in the treatment of microbial infections, particularly those of bacterial origin, including Staphylococcal origin. Ideally, the present invention is directed to a recombinant staphylococcal MSCRAMM or MSCRAMM-like proteins, or fragment thereof, with reduced binding to its host ligand, for use in therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/701,789, filed May 1, 2015, which is a divisional of U.S. patentapplication Ser. No. 12/865,336, which is the U.S. National Stage ofInternational Patent Application No. PCT/EP2009/051033, filed Jan. 29,2009, incorporated by reference herein in their entireties, and whichclaim priority to Ireland application No. 2008/0070, filed Jan. 31,2008, and Great Britain application No. 0801768.3, filed Jan. 31, 2008.

SEQUENCE LISTING

The following application contains a sequence listing in computerreadable format (CRF), submitted as a text file in ASCII format. Thecontent of the CRF is hereby incorporated by reference.

INTRODUCTION

The present invention relates to improved microbial antigen vaccines,pharmaceutical compositions, immunogenic compositions and antibodies andtheir use in the treatment of microbial infections, particularly thoseof bacterial origin, including Staphylococcal origin.

Multiple drug resistance (MDR) is an increasing problem amongst grampositive bacteria, particularly in hospitals. The widespread use ofantibiotics and other agents to treat bacterial infections has led tothe rapid development of bacteria resistant to the agents and manybacteria have multiple drug resistance. Thus, there is now a need toprovided improved therapies for dealing with such drug resistantinfections.

Staphylococci are Gram-positive bacteria of spherical shape, usuallyarranged in grape-like irregular clusters. Some are members of thenormal flora of the skin and mucous membranes of humans, others causesuppuration, abscess formation, a variety of pyogenic infections, andeven fatal septicaemia. Pathogenic staphylococci often hemolyze blood,coagulate plasma, and produce a variety of extracellular enzymes andtoxins.

The genus Staphylococcus has at least 30 species. The three main speciesof clinical importance are Staphylococcus aureus, Staphylococcusepidermidis, and Staphylococcus saprophyticus. Staphylococcus aureus iscoagulase-positive, which differentiates it from the other species. S.aureus is a major pathogen for humans. Almost every person has some typeof S. aureus infection during a lifetime, ranging in severity from foodpoisoning or minor skin infections to severe life-threateninginfections. The coagulase-negative staphylococci are normal human florawhich sometimes cause infection, often associated with implanteddevices, especially in very young, old and immunocompromised patients.Approximately 75% of the infections caused by coagulase-negativestaphylococci are due to S. epidermidis. Infections due toStaphylococcus warneri, Staphylococcus hominis, and other species areless common. S. saprophyticus is a relatively common cause of urinarytract infections in young women.

Staphylococci produce catalase, which differentiates them from thestreptococci. S. lugdunensis is also relevant in a clinical and ispresent in approximately 5 to 10% of cases of infective endocarditis.

S. aureus colonization of the articular cartilage, of which collagen isa major component, within the joint space appears to be an importantfactor contributing to the development of septic arthritis.Hematogenously acquired bacterial arthritis remains a serious medicalproblem. This rapidly progressive and highly destructive joint diseaseis difficult to eradicate. Typically, less than 50% of the infectedpatients fail to recover without serious joint damage. S. aureus is thepredominant pathogen isolated from adult patients with hematogenous andsecondary osteomyelitis.

In hospitalized patients, Staphylococcus bacteria such as S. aureus area major cause of infection. Initial localized infections of wounds orindwelling medical devices can lead to more serious invasive infectionssuch as septicaemia, osteomyelitis, mastitis and endocarditis. Ininfections associated with medical devices, plastic and metal surfacesbecome coated with host plasma and matrix proteins such as fibrinogenand fibronectin shortly after implantation. This ability of S. aureusand other staphylococcal bacteria to adhere to these proteins isessential to the initiation of infection. Vascular grafts, intravenouscatheters, artificial heart valves, and cardiac assist devices arethrombogenic and prone to bacterial colonization. Of the staphylococcalbacteria, S. aureus is generally the most damaging pathogen of suchinfections.

A significant increase in S. aureus isolates that exhibit resistance tomost of the antibiotics currently available to treat infections has beenobserved in hospitals throughout the world. The development ofpenicillin to combat S. aureus was a major advance in infection controland treatment. Unfortunately, penicillin-resistant organisms quicklyemerged and the need for new antibiotics was paramount. With theintroduction of every new antibiotic, S. aureus has been able to counterwith β-lactamases, altered penicillin-binding proteins, and mutated cellmembrane proteins allowing the bacterium to persist. Consequently,methicillin-resistant S. aureus (MRSA) and multidrug resistant organismshave emerged and established major footholds in hospitals and nursinghomes around the world (Chambers, H. F., Clin Microbiol Rev, 1:173,1988; and Mulligan, M. E., et al., Am J Med, 94:313, 1993). Today,almost half of the staphylococcal strains causing nosocomial infectionsare resistant to all antibiotics except vancomycin, and it appears to beonly a matter of time before vancomycin will become ineffective as well.

Thus, there remains a very strong and rapidly growing need fortherapeutics to treat infections from staphylococci such as S. aureuswhich are effective against antibiotic resistant strains of thebacteria.

In gram positive pathogens, such as Staphylococci, Streptococci andEnterococci, proteins, called adhesins, mediate such infections, forexample by promoting colonization, attachment to blood clots andtraumatized tissue. These specific microbial surface adhesins are termedMSCRAMMs (microbial surface components recognizing adhesive matrixmolecules) (Patti, J., et al., Ann Rev Microbiol, 48:585-617, 1994;Patti, J. and Hook, M., Cur Opin Cell Biol., 6:752-758, 1994). MSCRAMMsspecifically recognize and bind to extracellular matrix (ECM)components, such as fibronectin, fibrinogen, collagen, and elastin.These MSCRAMMs are found in many gram positive pathogens and their aminoacid sequences are related, they have similar modular design and commonbinding domain organization.

MSCRAMMs on the bacterial cell surface and ligands within the hosttissue interact in a lock and key fashion resulting in the adherence ofbacteria to the host. Adhesion is often required for bacterial survivaland helps bacteria evade host defence mechanisms and antibioticchallenges. Once the bacteria have successfully adhered and colonizedhost tissues, their physiology is dramatically altered and damagingcomponents such as toxins and enzymes are secreted. Moreover, theadherent bacteria often produce a biofilm and quickly become resistantto the killing effect of most antibiotics.

A bacterium can express MSCRAMMs that recognize a variety of matrixproteins. Ligand-binding sites in MSCRAMMs appear to be defined byrelatively short contiguous stretches of amino acid sequences (motifs).Because a similar motif can be found in several different species ofbacteria, it appears as though these functional motifs are subjected tointerspecies transfer (Patti and Hook, Cur Opin Cell Biol, 6:752-758,1994). In addition, a single MSCRAMM can sometimes bind several ECMligands.

MSCRAMMs can mediate infection by binding to proteins includingFibrinogen (Fg) and/or Fibronectin (Fn) etc. Fibrinogen and Fibronectinare proteins found in blood plasma and play key roles in hemostasis andcoagulation.

Fibrinogen is composed of six polypeptide chains, two Aα, two Bβ and twoγ-chains. The C-terminal part of the γ-chain is biologically importantand interacts with the platelet integrin during platelet adherence andaggregation. It is this region which is also targeted by Staphylococcusaureus resulting in Fibrinogen-dependant cell clumping and tissueadherence.

Staphylococcus aureus has several surface expressed proteins whichstimulate platelet activation and aggregation. The Staphylococcus aureusMSCRAMM proteins include but are not limited to the following:

-   -   Fibrinogen binding protein clumping factor A (ClfA);    -   Fibrinogen binding protein clumping factor B (ClfB);    -   Fibronectin-fibrinogen binding protein A (FnBPA);    -   Fibronectin-fibrinogen binding protein B (FnBPB); and    -   S. aureus surface proteins SasA, SasG, SasK etc.

Table 1 below outlines a selection of various Staphylococcus aureus cellwall-anchored surface proteins.

TABLE 1 Surface protein aa^(a) Ligand(s)^(b) Motif^(c) Sortase^(d)Protein A (Spa) 508 Immunoglobulin, von Willebrand LPETG A Factor,TNFR^(e) Fibronectin binding protein A 1,018 Fibronectin, fibrinogen,elastin LPETG A (FnbpA) Fibronectin binding protein B 914 Fibronectin,fibrinogen, elastin LPETG A (FnbpB) Clumping factor A (ClfA) 933Fibrinogen, complement factor I LPDTG A Clumping factor B (ClfB) 913Fibrinogen, cytokeratin 10 LPETG A Collagen adhesion (Cna) 1,183Collagen LPKTG A SdrC 947 Unknown LPETG A SdrD 1,315 Unknown LPETG ASdrE 1,166 Unknown LPETG A Pls 1,637 Unknown LPDTG A SasA 2,261 UnknownLPDTG A SasB 937 Unknown LPDTG A SasC 2,186 Unknown LPNTG A SasD 241Unknown LPAAG A SasE/IsdA 354 Heme^(f) LPKTG A SasF 637 Unknown LPKAG ASasG/Aap 1,117 Unknown^(g) LPKTG A SasH 308 Unknown LPKTG ASasI/HarA/IsdH 895 Haptoglobin LPKTG A SasJ/IsdB 645 Hemoglobin, hemeLPQTG A SasK 211 Unknown LPKTG A IsdC 227 Heme NPQTN B ^(a)aa, proteinlength in amino acids. ^(b)Molecular component(s) recognized and boundby protein. ^(c)Consensus motif recognized by sortase and present inC-terminal cell wall sorting signal. ^(d)Sortase for which cell wallsurface protein is substrate. ^(e)TNFR, tumor necrosis factor receptor^(f)also binds to proteins in desquamated epithelial cell. Promotesresistance to bactericidal lipids and lactoferrin ^(g)also binds todesquamated nasal epithelial cells. Involved in biofilm formation.

Other Staphylococcal bacteria express surface expressed proteins(MSCRAMMs) which are similar to the clumping factors or binding proteinslisted above. These include but are not limited to:

-   -   SdrF, SdrG and SdrH from S. epidermidis wherein SdrG/F have been        shown to bind fibrinogen and collagen.    -   Fbl from Staphylococcus lugdunensis is a fibrinogen-binding        protein. Fbl is a member of the Sdr-family, a group of        staphylococcal cell surface proteins containing a characteristic        serine-aspartate repeat region. The fibrinogen-binding domain of        Fbl has been mapped to 313 amino acids, and shows 62% identity        to the corresponding region in clumping factor A (ClfA) from        Staphylococcus aureus.

Other ligand-binding proteins/adhesins include Isd proteins(iron-regulated surface determinants), which although all of them arenot MSCRAMMs per se (e.g. IsdB and IsdH) promote adhesion of bacteria toextracellular matrix components and are referred to herein as“MSCRAMM-like proteins”. It is known that IsdA promotes adhesion tosquamous cells, and has weak affinity for fibrinogen and fibronectin, somay technically be defined as an MSCRAMM.

Clumping factor A (ClfA) was the first Fibrinogen γ-chain-binding S.aureus adhesin identified. Fibronectin-fibrinogen binding protein A(FnBPA) and Fibronectin-fibrinogen binding protein B (FnBPB) weresubsequently recognized as bi-functional proteins found to bind the sameC-terminal peptide segment in the γ-chain of Fg. ClfA and FnBPs havestructural features that are common to all cell-wall anchored proteinsexpressed in Gram-positive bacteria, including ClfB.

Clumping factor A (ClfA), for example, is a surface located protein ofStaphylococcus aureus. ClfA is an important virulence factor of S.aureus. It contributes to the pathogenesis of septic arthritis andendocarditis. ClfA is the archetype of a family of surface-associatedproteins with similar structural/modular organization, including but notlimited to ClfB, SdrD, SdrE etc,

ClfA contains a 520 amino acid N-terminal A domain (the FibrinogenBinding Region), which comprises three separately folded subdomains N1,N2 and N3. The A domain is followed by a serine-aspartate dipeptiderepeat region and a cell wall- and membrane-spanning region, whichcontains the LPDTG-motif for sortase-promoted anchoring to the cellwall. ClfA is present in practically all S. aureus strains (Peacock S J,Moore C E, Justice A, Kantzanou M, Story L, Mackie K, O'Neill G, Day N PJ (2002) Virulent combinations of adhesin and toxin genes in naturalpopulations of Staphylococcus aureus. Infect Immun 70:4987-4996). Itbinds to the C-terminus of the γ-chain of fibrinogen, and is therebyable to induce clumping of bacteria in fibrinogen solution (McDevitt D,Nanavaty T, House-Pompeo K, Bell E, Turner N, McEntire L, Foster T, HöökM (1997) Characterization of the interaction between the Staphylococcusaureus clumping factor (ClfA) and fibrinogen. Eur J Biochem 247:416-424and McDevitt D, Francois P, Vaudaux P, Foster T J (1994) Molecularcharacterization of the clumping factor (fibrinogen receptor) ofStaphylococcus aureus. Mol Microbiol 11:237-248).

3D Structural analysis of ClfA and the related fibrinogen-bindingproteins SdrG and ClfB has revealed that the ligand-binding A domain inall these related proteins are all composed of three subdomains N1, N2and N3, with residues 221-559 corresponding to Regions N2-N3 being thesmallest truncate that retains the ability to bind fibrinogen. It hasbeen found that amino acid residues 532 to 538 correspond to thelatching peptide region of ClfA. Each subdomain comprises nine β-strandsthat form a novel IgG-type fold. The fibrinogen γ-chain peptide-bindingsite in these proteins is located in a hydrophobic groove at thejunction between N2 and N3. It has been found that there is significantstructural similarity between the 3d structure of these proteins, thisis due to one or more of related amino acid sequence, similar modulardesign and common binding domain organization.

SdrC, SdrD, SdrE, FnBPA-A (all seven isoforms) and FnBPB-B (all sevenisoforms) have similar modular organization, thus using PHYRE molecularmodeling, these proteins would be expected to have the same 3Dstructure.

IsdA and IsdB do not have the same type of structure as Clf or Sdrproteins. They have a novel motif called NEAT which is involved inligand binding. However, the NEAT motif is similar to the 3D structureof Clf or Sdr, in that is composed of a sandwich of beta strands (betasandwich fold that consists of two five-stranded antiparallel betasheets) and is a member of the Ig-superfamily (Pilpa et al “SolutionStructure of the NEAT (NEAr Transported) Domain from IsdH/HarA: theHuman Hemoglobin Receptor in Staphylococcus aureus” J. Mol. Biol. (2006)360:435-447) The 3D structure of the NEAT motif of IsdH has been solvedand residues in loop 1b-2 predicted.

Expression of ClfA on S. aureus hampers phagocytosis by both macrophagesand neutrophils (Palmqvist N, Patti J M, Tarkowski A, Josefsson E (2004)Expression of staphylococcal clumping factor A impedes macrophagephagocytosis. Microb Infect 6:188-195 and Higgins J, Loughman A, vanKessel K P M, van Strijp J A G, Foster T J (2006) Clumping factor A ofStaphylococcus aureus inhibits phagocytosis by human polymorphonuclearleukocytes. FEMS Microbiol Lett 258:290-296). In neutrophils this is dueto both a fibrinogen-dependent mechanism and to a fibrinogen-independentmechanism. In contrast, platelets are activated by bacteria expressingClfA through its interaction with GPIIb/IIIa leading to aggregation.This is most efficiently executed when fibrinogen is present, but thereis also a fibrinogen-independent pathway for platelet activation(Loughman A, Fitzgerald J R, Brennan M P, Higgins J, Downer R, Cox D,Foster T J (2005) Roles of fibrinogen, immunoglobulin and complement inplatelet activation promoted by Staphylococcus aureus clumping factor A.Mol Microbiol 57:804-818 and O'Brien L, Kerrigan S W, Kaw G., Hogan M.,Penadés J., Litt D., Fitzgerald D. J., Foster T. J. & Cox D. (2002)Multiple mechanisms for the activation of human platelet aggregation byStaphylococcus aureus: roles for the clumping factors ClfA and ClfB, theserine-aspartate repeat protein SdrE and protein A. Mol Microbiol 44,1033-1044).

ClfA is a virulence factor for induction of septic arthritis in mice(Josefsson E., Hartford O., O'Brien L, Patti J M, Foster T (2001)Protection against experimental Staphylococcus aureus arthritis byvaccination with clumping factor A, a novel virulence determinant. JInfect Dis 184:1572-1580). In addition, elimination of ClfA togetherwith another fibrinogen binding protein ClfB protected against systemicinflammation at the early stages of infection (Palmqvist N, Foster T,Fitzgerald R, Josefsson E, Tarkowski A (2005) Fibronectin-bindingproteins and fibrinogen-binding clumping factors play distinct roles instaphylococcal arthritis and systemic inflammation. J Inf Dis191:791-798).

The Staphylococcus aureus fibrinogen binding protein ClfA has beenisolated and characterized and is the subject of, for example, U.S. Pat.Nos. 6,008,341 and 6,177,084.

ClfA and ClfB have an identical structural (3D) organization andapproximately 27% amino acid identity. FnBPA has an approximately 25%amino acid identity to ClfA.

At present there are no MSCRAMM based vaccines approved and on themarket. Veronate®, a donor-selected staphylococcal human immune globulinintravenous (IGIV) targeting ClfA and SdrG, performed poorly in phaseIII clinical trials and was withdrawn from trials. It is currently beingre-evaluated to determine whether it is a viable treatment forStaphylococcal infections.

WO 2005/116064 is directed to FnBPA, which is a multifunctional bindingprotein of S. aureus. The N-terminal A domain of FnBPA resembles ClfAand has been found to bind fibrinogen. However. the C-terminal BCDdomains of FnBPA bind fibronectin, hence, FnBPA is a bifunctionalMSCRAMM.

WO 2005/116064 is based on the finding that in the presence oftransglutaminase, covalent linkages are formed between the bacterialadhesin FnBPA and the host protein fibronectin, rendering theassociation much stronger and essentially irreversible. Fibrinogen is amajor component (˜3 mg/ml) in blood where it serves as the final targetof the coagulation cascade. Fibronectin is less abundant, ˜0.3 mg/ml orone molecule of Fn for every 10-15 of fibrinogen. Fibrinogen andfibronectin are not thought to be associated in blood where theycirculate independently.

Importantly, WO 2005/116064 specifically relates to FactorXIIIa-catalyzed covalent cross-linking. WO 2005/116064 isolates multiplemutants in a recombinant FnBPA where residues with positively chargedside chains (i.e. transglutaminase substrates) were altered.Furthermore, WO 2005/116064 is directed to mutants which have alteredcovalent fibronectin not fibrinogen binding properties only.Additionally, this document does not demonstrate experimentally whetherthe binding of the mutant protein to the ligand is reduced and does notprovide any supporting immunogenicity data.

Thus, in view of the prevalence of multiple drug resistance in grampositive bacteria and the lack of successful therapies and vaccines forthese multi-drug resistant bacteria, any alternative therapy which candeal with such bacterial infections without using antibiotics will be ofsignificant value.

Furthermore, any improvements in efficacy over any known treatments orvaccines will be of particular importance, especially in a clinicalsetting.

Thus, the present invention is directed to providing an alternative andimproved therapy for such treating such bacterial infections.

STATEMENT OF THE INVENTION

According to a first general aspect of the invention, there is provideda recombinant staphylococcal MSCRAMM or MSCRAMM-like protein, orfragment thereof, with reduced binding to its host ligand, for use intherapy.

According to a preferred embodiment, there is provided a recombinantstaphylococcal fibrinogen binding MSCRAMM protein, or fragment thereofcomprising at least part of the fibrinogen binding region, without theability to bind fibrinogen for use in therapy.

According to a second aspect of the invention, there is provided amethod of inducing an immune response in an individual and/or treating apatient having a microbial infection, comprising administering to theindividual a recombinant staphylococcal MSCRAMM or MSCRAMM-like protein,or fragment thereof, or vaccine comprising the recombinantstaphylococcal MSCRAMM or MSCRAMM-like protein, or fragment thereof,with reduced binding to its host ligand.

According to a third aspect of the invention, there is provided avaccine comprising a recombinant staphylococcal MSCRAMM protein, orfragment thereof, with reduced binding to its host ligand.

According to a fourth aspect of the invention, there is provided anantibody raised against a recombinant staphylococcal MSCRAMM orMSCRAMM-like protein, or fragment thereof, with reduced binding to itshost ligand, preferably in the form of a hyperimmune serum

According to a fifth aspect of the invention, there is provided animmunogenic pharmaceutical composition comprising a recombinantstaphylococcal MSCRAMM or MSCRAMM-like protein, or fragment thereof,with reduced binding to its host ligand and a pharmaceuticallyacceptable adjuvant.

DETAILED DESCRIPTION

In this specification, the terms “adhesin”, “MSCRAMM” and “cell-wallanchored proteins” will be understood to be interchangeable and coverall microbial derived ligand binding proteins. Ideally, these proteinsbind fibrinogen, heme or haemoglobin, haptoglobin-haemoglobin, haemin,collagen and other such ligands. The term “MSCRAMM-like” proteins areintended to cover proteins or adhesins which have related amino acidsequences, similar modular design and/or common/similar binding domainorganization to such MSCRAMM proteins, such as Isd proteins. Ideally,the MSCRAMM-like proteins have similar binding domainorganization/modular design. Additionally, the MSCRAMM-like proteins mayhave at least 50%, preferably 60%, preferably 75%, more preferably 85%,even more preferably 95%, still more preferably 99% or more amino acidsequence identity with the MSCRAMM proteins.

It will also be understood that any of the percentage identities orhomologies referred to in the specification are determined usingavailable conventional methods over the entire/whole length of thesequence.

The term “micro-organism”, “microbe”, “microbial” or the like includesbut is not limited to organisms including bacteria, fungi, viruses,yeasts and/or moulds.

The term “immunologically effective amount” covers those amounts whichare capable of stimulating a B cell and/or T cell response.

According to a first general aspect of the invention, there is provideda recombinant staphylococcal MSCRAMM or MSCRAMM-like protein, orfragment thereof, comprising at least part of the ligand binding region,with reduced binding to its host ligand, for use in therapy. Such arecombinant protein may be used in the treatment of microbialinfections, such as the treatment of sepsis, septic arthritis and/orendocarditis or other similar conditions or disease states. Suchmicrobial infections may ideally be caused by Staphylococci or othersimilar micro-organisms.

According to one particular embodiment of this aspect of the invention,the recombinant MSCRAMM or MSCRAMM-like protein, or fragment thereof,has reduced or lacks the ability to non-covalently bind its host ligand.

Thus, it will be understood that the recombinant staphylococcal MSCRAMMor MSCRAMM-like protein, or fragment thereof, may have reduced bindingwith its host ligand or binding with the host ligand may be prevented.

It is postulated, according to the invention, that the non-covalentbinding that takes place during binding, via Dock, Lock and Latching(DLL), of the MSCRAMM or MSCRAMM-like protein to its ligand may bereduced or prevented. It is established that the first step in bindingof an MSCRAMM to its ligand involves a non-covalent interaction via theDLL model. These are the primary non-covalent MSCRAMM interactions withthe ligand. The final stages in MSCRAMM-ligand binding involve covalentinteractions. In this particular embodiment, the recombinant MSCRAMM orMSCRAMM-like protein, or fragment thereof, has reduced or lacks theability to non-covalently bind its host ligand due to altered dock, lockand latching. One or more of the dock, lock or latching steps may bealtered.

The DLL model was elucidated from the 3D structure of SdrG in complexwith its ligand. ClfA has now been shown to act by a minor variation ofthe DLL mechanism (Ganech et al (2008) “A structural model of theStaphylococcus aureus Clfa-fibrinogen interaction opens new avenues forthe design of anti-staphylococcal therapeutics”. PloS Pathog 4(11);e1000226). The DLL model specifically relates to the non-covalentinteractions involved in ligand binding. The DLL model is inferred forall other proteins of similar structural type (whether by amino acidsimilarity/homology or structural organization homology), including butnot limited to MSCRAMM or MSCRAMM-like proteins.

In relation to MSCRAMMs ClfA/ClfB in particular, it has been found thatthe minimal ligand binding domain comprises Region A subregions N1 toN3, specifically subregions N2 and N3 which comprise a variant Dev-IgGIg fold. The variant Dev-IgG Ig fold is new variant of theimmunoglobulin motif also called the DE-variant. It is postulated that ahydrophobic pocket formed between the two DEv-IgG domains of ClfA/B isthe ligand-binding site for the fibrinogen γ-chain. Essentially, theligand binds to the hydrophobic groove separating N2 and N3.Specifically, during ligand binding the unfolded peptide component ofthe ligand inserts into the groove located between the N2 and N3subdomains. The latching peptide at the C-terminus of subdomain N3undergoes a conformational change and inserts between two beta strandsin subdomain N2, thus, locking the ligand in place. Indeed, mutagenicsubstitution of residues Tyr256, Pro336, Tyr338 and Lys389 in theclumping factor, which are proposed to contact the terminal residues⁴⁰⁸AGDV⁴¹¹ of the fibrinogen γ-chain, resulted in proteins with no ormarkedly reduced affinity for fibrinogen. Further details of thisspecific embodiment are expanded on later.

Whilst these teachings relate to clumping factors, ClfA in particular,they are equally applicable to other MSCRAMMs and/or MSCRAMM-likeproteins, which have similar modular binding domain organization andbind ligands in similar ways.

Thus, in order to provide recombinant staphylococcal MSCRAMM orMSCRAMM-like proteins, or fragment thereof, with reduced binding to itshost ligand, the full length protein, ligand binding domain, minimalligand binding domain or fragment thereof may be altered to reduce orprevent binding to its host ligand. Ideally, for ClfA/ClfB and othersimilar MSCRAMM or MSCRAMM-like proteins, Region A subregion N2 and N3,which ideally comprise a variant Dev-IgG Ig fold, may be altered toprevent or reduce binding to its host ligand. Such an alteration isdesigned to prevent the ligand binding to the hydrophobic grooveseparating minimal ligand binding domains needed for DLL.

Such alterations in the ligand binding domain may take place at theamino acid level, by amino acid substitution or deletion, using eitherthe full length protein, ligand binding domain, minimal ligand bindingdomain or fragment thereof. It will be understood that proteins orfragments thereof with sufficiently high homology to the ligand bindingprotein may also be used. High homology as defined herein occurs when atleast 50%, preferably 60%, preferably 70%, preferably 80%, morepreferably 90%, even more preferably 95%, still more preferably 95% to99%, still more preferably 99% or more of the nucleotides or match overthe entire length of the DNA sequence or when used in connection withamino acid sequences when the amino acid sequences are not identical butproduce a protein having the same functionality and activity. It will beunderstood that these comments about high homology may also relate tothe 3D structure of the protein, i.e. modular binding domainorganization.

It will be understood that the complete ligand binding protein, theligand binding domain, the minimal ligand binding domain or a fragmentthereof may be used.

The use of truncated proteins of the ligand binding protein such as theligand binding domain, the minimal ligand binding domain, or the use offragments thereof is advantageous for ease of manufacture and overcomingother problems such as unwanted cleavage of the protein. For example,the latching peptide, present in the minimal ligand binding domain, maybe deleted/removed or altered. For example, the latching peptide in ClfAcorresponds to Region A amino acids 532 to 538 and in ClfB to Region Aamino acids 530-540 (Walsh et al (2004) JBC 279(49): 50691-50699). Theseresidues may be altered, substituted or removed/deleted in order toprevent the ligand binding to the MSCRAMM via DLL. In this way the DLL“latching” of the MSCRAMM to its ligand is prevented. This “latching”occurs by way of a non-covalent interaction. In one embodiment, thelatching peptide is removed in its entirety along with the remainingRegion A C-terminal amino acid residues. According to anotherembodiment, the latching peptide region only is removed. According toyet another embodiment, the latching peptide region undergoes amino acidsubstitution to result in the reduction or prevention of ligandbinding/latching. These comments are applicable to all MSCRAMM orMSCRAMM-like proteins with bind ligands by the DLL or similar models.

By altering the MSCRAMM or MSCRAMM-like protein in this manner, it ispossible to provide a ligand binding protein without the ability to bindits ligand, which stimulates a greater immune response upon immunizationthan the wild type protein. Advantageously, this reduces systemicinflammation, thereby decreasing microbial virulence. Consequently, thisaltered ligand binding MSCRAMM or MSRAMM-like protein which lacks theability to bind its ligand can be advantageously used in the treatmentof microbial infections. Thus, these findings present a new and valuablevaccine/immunization therapeutic against bacterial infections whichprovides better results when compared to a vaccine or immunizationtherapeutic derived from the wild type protein.

According to one embodiment of the invention the ligand is heme,haemoglobin or fibrinogen. Other ligands may be contemplated such ashaptoglobin-haemoglobin, haemoglobin, haemin, collagen etc.

According to another embodiment of the invention, the recombinantMSCRAMM protein is selected from

-   -   a fibrinogen binding protein; or    -   SdrD, SdrE, SdrG and/or SdrF.

Fibrinogen binding proteins have been expanded on above, and include butare not limited to ClfA, ClfB, FnBPA, FnBPB, Fbl, IsdA etc. It has beenshown that SdrG/F bind collagen. Other MSCRAMMs include SasA, SasG, SasKand SdrH.

The recombinant MSCRAMM-like protein may be selected from

-   -   IsdA, IsdB, and/or IsdH.

Based on the findings from fibrinogen binding MSCRAMM ClfA, similarnon-ligand binding mutants can be generated in for example the NEAT(NEAr Transporter) motif of Isd proteins including IsdH and IsdB. Asexpanded on above IsdA and IsdB do not have the same type of structureas the Clf or Sdr proteins. However, the NEAT motif in Isd is directlyinvolved in ligand binding (haptoglobin-haemoglobin, haemoglobin,haemin), thus, alterations in the NEAT motif will prevent thehost-ligand interaction in the same way as altering the DLL or DLL likehost-ligand interaction of Clf or Sdr. Many NEAT domain-containingproteins, including IsdA in Staphylococcus aureus, are implicated inhaem binding. It is postulated that the haem-binding property of IsdA iscontained within the NEAT domain. Crystal structures of the apo-IsdANEAT domain and in complex with haem have revealed a clathrinadapter-like β-sandwich fold with a large hydrophobic haem-bindingpocket. IsdB has two NEAT motifs and IsdA has one NEAT motif. Non-ligandbinding mutants of Isd proteins may be isolated by altering the residuespredicted for ligand binding, for example, by altering the residuesbetween beta strands and/or hydrophobic pocket. Additionally, the NEATmotif may be altered to effect non-covalent host-ligand interactions.

According to another embodiment of this aspect of the invention, thereis provided a method of inducing an immune response in an individualand/or treating a patient having a microbial infection, comprisingadministering to the individual a recombinant staphylococcal MSCRAMM orMSCRAMM-like protein, or fragment thereof, or vaccine comprising therecombinant staphylococcal MSCRAMM or MSCRAMM-like protein, or fragmentthereof with reduced binding to its host ligand.

According to another embodiment of this aspect of the invention, thereis provided a vaccine comprising a recombinant staphylococcal MSCRAMM orMSCRAMM-like protein, or fragment thereof, with reduced binding to itshost ligand.

According to another embodiment of this aspect of the invention, thereis provided an antibody raised against a recombinant staphylococcalMSCRAMM or MSCRAMM-like protein, or fragment thereof, with reducedbinding to its host ligand, preferably in the form of a hyperimmuneserum

According to another embodiment of this aspect of the invention, thereis provided an immunogenic pharmaceutical composition comprising arecombinant staphylococcal MSCRAMM or MSCRAMM-like protein, or fragmentthereof, with reduced binding to its host ligand

According to a preferred embodiment of the invention, there is provideda recombinant staphylococcal fibrinogen binding protein, or fragmentthereof comprising at least the part of fibrinogen binding region,without the ability to bind fibrinogen for use in therapy.

It will be understood that the recombinant Staphylococcal fibrinogenbinding protein, or fragment thereof, may be used in the treatment ofmicrobial infections, preferably Staphylococci infections such as in thetreatment of sepsis, septic arthritis and/or endocarditis or othersimilar conditions or disease states.

The fibrinogen binding region of the protein is altered so that it nolonger binds fibrinogen. As stated above, the alteration may take placeat the nucleotide or amino acid level. It will be understood thatproteins or fragments thereof with sufficiently high homology to thefibrinogen binding protein may also be used. High homology as definedherein occurs when at least 50%, preferably 60%, preferably 70%,preferably 80%, more preferably 90%, even more preferably 95%, stillmore preferably 95% to 99%, still more preferably 99% of the nucleotidesmatch over the entire length of the DNA sequence or when used inconnection with amino acid sequences when the amino acid sequences arenot identical but produce a protein having the same functionality andactivity. It will be understood that these comments about high homologymay also relate to the 3D structure of the protein.

It will be understood that the complete fibrinogen binding protein, thefibrinogen binding region, the minimal fibrinogen binding region, or afragment thereof may be used. The use of truncated proteins or fragmentsthereof is advantageous for ease of manufacture and overcoming otherproblems such as unwanted cleavage of the protein. This is expanded onbelow.

Such fragments should ideally comprise at least part of the fibrinogenbinding region of the MSCRAMM. The advantages of using a truncatedprotein or fragment thereof of the, comprising for example one or moresubdomains of the ligand-fibrinogen binding region only, relate to theability to purify the protein at high yields without degradation. TheClfA protein fibrinogen binding region, otherwise referred to as the ARegion, comprises 3 subregions, N1, N2 and N3. Thus, the immunogenicfragment may comprise subregions N1, N2 and/or N3 of the ClfA A Regionor a fragment thereof. Thus, for example, in relation to ClfA, thefragment may comprise one or more of subdomains of Region A, N1, N2 orN3. Ideally, N2 and N3 may be used as this truncate is less likely toundergo proteolysis (a protease cleavage site has been reported betweenN1 and N2 in ClfA and ClfB) and can be expressed at higher levels in E.coli. N2 and N3 are the minimal fibrinogen binding region of Clfproteins.

It will be understood that although the following discussion relates tothe fibrinogen binding protein ClfA, that these comments are equallyapplicable to other MSCRAMMs, MSCRAMM-like proteins and in particularother fibrinogen binding proteins which are structurally similar eitherat an amino acid or protein structure level to ClfA, for example asClfB, Fbl and SdrF/G (which also bind collagen). Furthermore, theseteachings are applicable to FnBPA and FnBPB. Thus, although thefollowing comments relate to fibrinogen binding proteins, they areequally applicable to other MSCRAMM or MSCRAMM-like proteins which bindligands other than fibrinogen.

We have unexpectedly found that this altered fibrinogen binding protein,truncate or fragment thereof, without the ability to bind fibrinogenstimulates a greater immune response upon immunization than the wildtype protein which binds to fibrinogen in the normal manner.Advantageously, this altered fibrinogen binding protein does not provokesystemic inflammation when expressed by S. aureus, thus, microbialvirulence is decreased. Consequently, this altered protein which lacksthe ability to bind fibrinogen can be advantageously used in thetreatment of microbial infections. We have also found contrary toexpectations that the protection effect of the altered fibrinogenbinding protein is greater than the wild type protein. We have foundthat a pharmaceutical composition or vaccine comprising such an alteredrecombinant protein is more effective than a pharmaceutical compositionor vaccine comprising the same recombinant protein in an unaltered (wildtype) form, such as ClfA, ClfB, SdrG etc.

Thus, these findings present a new and valuable vaccine/immunizationtherapeutic against bacterial infections which provides better resultswhen compared to the wild type protein when also used as avaccine/immunization therapeutic.

It will be understood that the altered protein, whether MSCRAMM orMSCRAMM-like or fibrinogen or other ligand binding, may be used in thegeneration of antibodies, including monoclonal, polyclonal, chimeric,humanized antibodies or fragments thereof, for use in the treatment ofsuch microbial infections. Compositions may then be provided whichinclude such antibodies, such as a hyperimmune serum, and thesecompositions may be used in the treatment of patients infected withStaphylococcus infections.

Thus, the proteins or active fragments thereof may be used to inhibitthe binding of Staphylococci to the extra-cellular matrix (ECM) and toprevent/treat Staphylococci infections in a patient.

Furthermore, the proteins or active fragments thereof, and antibodies tothe proteins are useful in the treatment of infections fromStaphylococcal infections, for the development of vaccines for active orpassive vaccination, and when administered as a pharmaceuticalcomposition to a wound or a medical device, both the proteins andantibodies are useful as blocking agents to prevent microbial infection.For example, these proteins or fragments thereof may be used in activevaccines, and the antibodies to these proteins in passive vaccines.

These vaccines and products described herein present a significantimprovement over the prior art, which teaches the general use ofMSCRAMMs to impart immunization, but does not teach the unexpected andimproved vaccines or products described herein.

The preparation of proteins, DNA and antibodies are well known in theart and will not be described in detail herein. Conventional techniquesare ideally used in the generation of these molecules. The inventionwill also be understood to cover nucleic acid constructs containing thenucleic acid or amino acid sequence of interest, recombinant host cellscontaining such nucleic acid constructs to express the protein ofinterest, and immunogenic compositions.

For administration, the protein composition may be dispersed in asterile, isotonic saline solution or other pharmaceutically acceptableadjuvant.

It will be understood that the vaccine may be a DNA or protein vaccine.

Immunization may take place by the injection of DNA, protein orantibodies. Alternatively, an attenuated live organism that includes andexpresses the DNA may be administered.

The amount of DNA, protein or antibodies that may be administered willdepend on several mitigating factors, including dependence on thepromoter strength, protein expression and immunogenicity of theexpressed gene. These may be altered for each new application to obtainthe desired immunologically effective amount required.

According to another embodiment of this invention, there is provided amethod of inducing an immune response in an individual and/or treating apatient having a microbial infection, comprising administering to theindividual a recombinant Staphylococcal fibrinogen binding protein, orfragment thereof comprising at least the fibrinogen binding region,without the ability to bind fibrinogen.

According to further preferred embodiment of the invention, there isprovided a vaccine comprising a recombinant Staphylococcal fibrinogenbinding protein, or fragment thereof comprising at least part of thefibrinogen binding region, without the ability to bind fibrinogen.

According to a still further preferred embodiment of the invention,there is provided an antibody raised against a recombinantStaphylococcal fibrinogen binding protein, or fragment thereofcomprising at least part of the fibrinogen binding region, without theability to bind fibrinogen, preferably in the form of a hyperimmuneserum.

According to a yet further preferred embodiment of the invention, thereis provided an immunogenic pharmaceutical composition comprising arecombinant Staphylococcal fibrinogen binding protein, or fragmentthereof comprising at least part of the fibrinogen binding region,without the ability to bind fibrinogen and a pharmaceutically acceptableadjuvant.

Ideally, the recombinant Staphylococcal fibrinogen binding protein orfragment thereof is derived from S. aureus, S. epidermidis and/or S.lugdunensis.

The fibrinogen binding protein of these embodiments may be selected fromone of the following Fbl, SdrF, and/or SdrG (which are also collagenbinding). Alternatively, the fibrinogen binding protein may be selectedfrom one of the following Fibrinogen binding protein clumping factor A(ClfA), Fibrinogen binding protein clumping factor B (ClfB),Fibronectin-fibrinogen binding protein A (FnBPA), Fibronectin-fibrinogenbinding protein B (FnBPB). IsdA promotes adhesion has weak affinity forfibrinogen and fibronectin, so may technically be defined as afibrinogen binding MSCRAMM.

It will be understood that nucleotide or amino acid substitutions ordeletions within the fibrinogen binding region of such fibrinogenbinding proteins result in a recombinant protein without the ability tobind fibrinogen.

ClfA-fibrinogen binding has been elucidated to occur by a dock, lock andlatch (DLL) mechanism similar to that of SdrG. The DLL model wasexpanded on above. Region A of ClfA is responsible for theprotein-ligand interaction. As shown in FIG. 11, the modular structureof several fibrinogen binding MSCRAMM are similar and all contain RegionA similar to ClfA.

The fibrinogen γ-chain peptide-binding site is located in a hydrophobicgroove at the junction between N2 and N3 of ClfA. Thus, thesubstitutions or deletions mentioned above are designed to alter theMSCRAMM protein-ligand interaction and prevent the non-covalent bindingof ClfA to fibrinogen.

According to one specific embodiment of the present invention, therecombinant Staphylococcal fibrinogen binding protein is a fibrinogenbinding-deficient mutant of ClfA. In this embodiment, Fibrinogen BindingRegion A of ClfA is altered by any means (such as substitution ordeletion mutations) so that it no longer binds fibrinogen.

Ideally, the fibrinogen binding protein is ClfA, however, ClfA bears 3Dstructural similarity to many other fibrinogen binding proteins. Thus,it will be understood that these comments relating to ClfA are equallyapplicable to other MSCRAMM fibrinogen binding proteins, including ClfB,FnBPA, FnBPB, Fbl, SdrG/F, IsdA etc. All of these proteins have similar3D structures, thus, similar alterations/mutations to the fibrinogenbinding region can be made to achieve the same results.

ClfA is a 993 amino acid protein, comprising a 520 amino acid fibrinogenbinding domain (from amino acids 40 to 559). This fibrinogen bindingdomain is the N Terminal A domain comprising subregions N1, N2 and N3.The entire fibrinogen region spanning N1 to N3 from amino acid 40 toamino acid 559 may be used in the invention. Alternatively, a truncateof the N1 to N3 region may be used, e.g. 221 to 559 (the minimalfibrinogen binding region), 221 to 531 (the minimal fibrinogen regionwithout the latching peptide and following residues) etc. Ideally,subregions N2 and N3, the minimal fibrinogen binding region, may be usedwhich correspond to amino acid residues 221 to 559. Alternatively, afragment of these subregions may be used.

It has been established that amino acid residues 221 to 559, coveringthe N2 and N3 regions, of ClfA play an important part in the binding tofibrinogen and are the minimal fibrinogen binding region. We also haveunexpectedly found that mutation of amino acid residues in this regionresults in an expressed protein which can be recognized by the hostimmune defences but lacks fibrinogen binding and hence, reduces theassociated virulence. This region (the 339 amino acid fibrinogen bindingdomain) of ClfA has a specific 3D structure, a so-called DE-variant IgGfold, and is the minimum Fg-binding truncate which if altered (viasubstitution or deletion etc) can provide an improved therapy.

The alteration to result in the loss of fibrinogen binding activity maytake place by substitution, addition or insertion or deletion at eitherthe nucleotide or amino acid level. Ideally, the substitution negativelyaffects the 3D structure (e.g. of the a so-called DE-variant IgG fold)of the protein or fragment so it can no longer bind fibrinogen.

Ideally, the nucleotide or amino acid substitution reduces thenon-covalent interaction with fibrinogen, preferably by preventingligand binding to the hydrophobic pocket separating N2 and N3 of RegionA of the fibrinogen binding protein. Alternatively, the latching peptideregion corresponding to amino acids 532 to 538 may be altered bysubstitution or deleted to prevent ligand binding. Additionally, atruncate/fragment lacking the latching peptide region and optionally theremainder of the C-terminal protein residues, i.e. lacking amino acidresidues 532 to 559, may be used.

According to one specific embodiment of this aspect of the invention,the fibrinogen binding-deficient mutant of ClfA may be constructed byexchanging amino acids P₃₃₆ for serine and/or Y₃₃₈ for aspartate,respectively. The choice of residues was based on the X-ray crystalstructure of ClfA and the observation that individual changes to theproline or the tyrosine reduced binding affinity. Surprisingly, we foundthat this mutant ClfA protein (rClfAP₃₃₆ S Y₃₃₈A) stimulated an immuneresponse and can be used in the generation of a much more effectivevaccine or antibody therapy. This substitution may take place in thefull length fibrinogen binding protein, the fibrinogen binding region,the minimal fibrinogen binding region, or a fragment thereof.

According to another specific embodiment of this aspect of theinvention, the fibrinogen binding-deficient mutant of ClfA may beconstructed by exchanging amino acids P₃₃₆ for aspartate and/or Y₃₃₈ forserine, respectively. As with the previous embodiment, this mutant ClfAprotein (rClfAP₃₃₆ A Y₃₃₈S) can also be used in the generation of a muchmore effective vaccine or antibody therapy.

Alternatively, the alteration may be in the form of a deletion,comprising the fibrinogen binding region without the latching peptidesequence (amino acids 532 to 538), to result in a recombinant fibrinogenbinding protein without the ability to non-covalently bind fibrinogen.In this embodiment, amino acid residues 221 to 531 of Region A of ClfAare used, which lack the latching peptide and following C-terminalresidues. Alternatively, an amino acid substitution in the latchingpeptide amino acids 532 to 538 which prevents the DLL of the fibrinogenmay be contemplated.

It is understood that all proteins in the Clf-Sdr family binds ligandsby the DLL model. By modelling the 3D structure, it is possible topredict the latching peptide and make a truncate that lacks it, eitherin the full length (N1 to N3) or the minimal ligand binding truncateN2-N3, or a fragment thereof.

We found that these substitution rClfA proteins (whether deletionmutants, substitutions or truncates) reduced virulence and diseaseoutcome, and surprisingly induced less systemic inflammation that thewild type protein.

Thus, immunization with these mutant proteins is expected to, based onthe proteins tested, enhance the level of antibodies which recognizedboth the mutant and wild type protein and to provide for a greaterimmune response than the wild type protein.

Thus, ClfA which has been altered so that it no longer binds fibrinogenis a useful therapeutic candidate for active or passive immunization. Inthis way, the altered ClfA protein itself may be used as a vaccine orantibodies raised to this altered ClfA protein may be used. As above thevaccine may be a DNA or protein vaccine.

The following sequences outlined in the table below may be used inaccordance with the invention.

SEQ ID No Description Length A Region 1 wt rClfA-full length aa sequence933 aa — (Example 1) 2 wt rClfA A Region-full length DNA sequence 1560N1 to N3 (Example 1) nucleotides 3 wt rClfA A Region-full length aasequence 520 aa N1 to N3 (Example 1) 4 rClfAPYI A Region 520 aa N1 to N3(Example 1) 5 rClfAPYII A Region 520 aa N1 to N3 (Example 1) 6 wt rClfAA Region-full length aa sequence with 530 aa N1 to N3 additional N and Cterminal residues¹ (Example 2) 7 rClfAPYI A Region with additional N andC 530 aa N1 to N3 terminal residues¹ (Example 1) 8 rClfAPYII A Regionwith additional N and C 530 aa N1 to N3 terminal residues¹ 9 rClfA221-559 (Example 2) 339 aa N2 and N3 10 rClfA 221-559 with additional Nand C terminal 349 aa N2 and N3 residues¹ (Example 2) 11 rClfA PY221-559 339 aa N2 and N3 (Example 2) 12 rClfA PY 221-559 with additionalN and C 349 aa N2 and N3 terminal residues¹ (Example 2) 13 rClfA 221-531(delta latch truncate) with 321 aa N2 and N3³ additional N and Cterminal residues ² (Example 2) 14 rClfAPY 221-531 (delta latchtruncate) 311 aa N2 and N3³ ¹Additional N residues (N-terminal extension(6 × His tag and additional residues) comprise 6 His residues, followedby Gly and Ser. Additional C terminal residues comprise Lys followed byLeu (other additional N and C terminal residues may be used-depending onthe primer used or N/C terminal tags required) ² Additional N residues(6 × His tag and additional residues) comprise 6 His residues, followedby Gly and Ser. Additional C terminal residues comprise Arg followed bySer (other additional N and C terminal residues may be used-depending onthe primer used or N/C terminal tags required)) ³without the latchingpeptide corresponding to aa residues 532 to 538 and remainder A RegionC-terminal residues i.e. lacking amino acid residues 532 to 559.

Ideally, the recombinant Staphylococcal fibrinogen binding proteincomprises the amino acid sequence according to any of SEQ ID Nos. 1 to 3wherein residue P₃₃₆ and/or Y₃₃₈ are substituted with either serineand/or alanine, or a fragment thereof.

Alternatively, the fragment of the recombinant Staphylococcal fibrinogenbinding protein comprises the amino acid sequence according to any ofSEQ ID No. 4 to SEQ ID No. 14. SEQ ID NOs 4 and 5 correspond to the ClfAA domain N1, N2, N3 only, rClfA P₃₃₆S Y₃₃₈A and rClfA P₃₃₆A Y₃₃₈Srespectively as outlined in the table above.

It is also postulated, based on the substitutions in the latch whichwere made in SdrG, that substitutions in the latch that are defective inthe conformational change or beta strand complementation will also bedefective in ligand binding. Thus, ideally, the substitutions are inamino acid residues 532 to 538 which correspond to the latching peptideand affect the ability of the peptide to undergo conformational change,or bind the ligand or both. Alternatively, the alteration may compriseremoving the amino acid residues 532 to 538 (delta latch peptide)altogether, to give similar results. Additionally, a C-terminaltruncation mutant lacking amino acid residues 532 to 559 (including thelatching peptide residues) will also effect binding to the ligand.

However, it will also be contemplated that other amino acid residuescould be substituted other than those specifically recited above. Forexample, Glu 526, Val 527, Tyr 256 and Lys 389 may be substituted toalter the fibrinogen binding properties of the protein or fragmentthereof. Thus, any substitution which reduces binding ability may becontemplated. Ideally, such substitutions or deletions effect thehydrophobic pocket and associated mechanism for binding the ligand inthe hydrophobic trench such as homologues Val527 in ClfA and N526 inClfB. In ClfB, Q235 and N526 have been studied to shown to reducebinding. A similar study was done with FnBPA where N304 and F306 wereshown to be important for Fg binding. Thus, mutations in these aminoacid residues will affect ligand binding.

It will be understood that these comments are equally applicable toother fibrinogen binding proteins, such as ClfB, SdrG, FnBPA, FnBPB.Thus, the treatment (vaccine, antibody or pharmaceutical compositionetc) may comprise the complete Fibrinogen Binding Region or a fragmentthereof.

In the specification, the terms “comprise, comprises, comprised andcomprising” or any variation thereof and the terms “include, includes,included and including” or any variation thereof are considered to betotally interchangeable and they should all be afforded the widestpossible interpretation.

The invention is not limited to the embodiment hereinbefore described,but may be varied in both construction and detail within the scope ofthe claims.

The present invention will now be described with reference to thefollowing non-limiting figures and examples.

FIGS. 1 to 15 show the results of Example 1.

FIG. 1 shows the severity of arthritis (A), measured as arthritic index,and weight loss (B) in mice inoculated with S. aureus strain Newman, andclfAPYI, clfAPYII, and clfA null mutants. 3.2×10⁶-6.0×10⁶ cfu of S.aureus strains were inoculated. Data are presented as medians (squaresor center lines), interquartile ranges (boxes), and 80% central ranges(whiskers). Data from three experiments are pooled. N_(Newman)=27-30,N_(clfAPYI)=30, N_(clfAPYII)=10, and N_(clfA)=16-20.

FIG. 2 shows the bacterial growth in kidneys in mice 7-8 days afterinoculation with 3.2×10⁶-6.0×10⁶ cfu of S. aureus strain Newman, andclfAPYI, clfAPYII, and clfA null mutants. Data are presented as cfu perkidney pair. Where no growth was detectable, the count was put tohighest possible count according to what dilution was used. Data fromthree experiments are pooled. N_(Newman)=26, N_(clfAPYI)=30,N_(clfAPYII)=10, and N_(clfA)=15.

FIG. 3 shows the survival of mice after inoculation with 5.2, 5.1 or3.3×10⁷ cfu of S. aureus strain Newman, clfAPYI mutant or clfA nullmutant, respectively. N=10 per group from start.

FIG. 4 shows the survival of mice after inoculation with 9.4, 7.9, 10.7or 9.8×10⁶ cfu of S. aureus strain LS-1, and clfAPYI, clfAPYII or clfAnull mutants, respectively. N=15 per group from start.

FIG. 5 shows the survival of mice immunized with BSA, recombinant ClfAor recombinant ClfAPY (i.e. ClfAPYI recombinant protein A domain) andinoculated with 2.3×10⁷ cfu of S. aureus Newman. N=15 per group fromstart.

FIG. 6 shows the frequency of arthritic mice inoculated with3.2×10⁶-6.0×10⁶ cfu of S. aureus strain Newman wild-type, and clfAPYI,clfAPYII, and clfA null mutants. Data from three experiments are pooled.N_(Newman)=27-30, N_(clfAPYI)=30, N_(clfAPYII)=10, and N_(clfA)=16-20.

FIG. 7 shows the severity of arthritis measured as arthritic index inmice inoculated with 5.2, 5.1 or 3.3×10⁷ cfu of S. aureus strain Newmanwild-type, clfAPYI mutant or clfA null mutant, respectively. Data arepresented as medians (squares), interquartile ranges (boxes), and 80%central ranges (whiskers). N_(Newman)=0-10, N_(clfAPYI)=9-10, andN_(clfA)=0-10.

FIG. 8 shows the weight loss in mice inoculated with 5.2, 5.1 or 3.3×10⁷cfu of S. aureus strain Newman wild-type, clfAPYI mutant or clfA nullmutant, respectively. Data are presented as medians (center line),interquartile ranges (boxes), and 80% central ranges (whiskers).N_(Newman)=0-10, N_(clfAPYI)=9-10, and N_(clfA)=0-10.

FIG. 9 shows the severity of arthritis measured as arthritic index inmice immunized with BSA, recombinant ClfA or recombinant ClfAPY (i.e.ClfAPYI recombinant protein A domain) and inoculated with 4.0×10⁶ cfu ofS. aureus Newman. Data are presented as medians (squares), interquartileranges (boxes), and 80% central ranges (whiskers). N_(BSA)=14,N_(clfAPY)=14, and N_(clfA)=15 per group from start.

FIG. 10 gives the nucleotide and amino acid sequence of wild-type ClfA Adomain protein (rClfA), domains N123 only, with the residues highlightedwhich are altered in the following examples (P₃₃₆ and Y₃₃₈) to give riseto rClfAPYI/II (SEQ ID No.3). It is this recombinant protein A domainwhich was used in vaccination in the following examples.

FIG. 11 shows an illustrative representation of the structure of FnBPA,ClfB, ClfA and SdrG proteins. Region A is the fibrinogen binding region,S is the signal sequence, W is the cell wall spanning domain, M is themembrane anchor including the LPXTG motif, + represent positivelycharged residues and R is the repeat region. In ClfA Region A comprisesN123 (not shown). The BCD region of FnBPA (and the shorter CD region ofFnBPB—not shown) binds fibronectin.

FIG. 12 shows the specific antibody responses to recombinantClfAPY40-559 in serum samples of mice immunized with bovine serumalbumin (BSA), recombinant ClfA40-559 (rClfA), or recombinantClfAPY40-559 (rClfAPY), 9 days after the second booster immunization,which was one day before infection with 2.3×10⁷ cfu/mouse of S. aureusstrain Newman wildtype for induction of sepsis. Data are presented asmedians (center lines), interquartile ranges (boxes), and 80% centralranges (whiskers). N_(BSA)=13-15, N_(rClfA)=15, and N_(rClfAPY)=15.

FIG. 13 shows the specific antibody responses to recombinant ClfA40-559in serum samples of mice immunized with bovine serum albumin (BSA),recombinant ClfA40-559 (rClfA), or recombinant ClfAPY40-559 (rClfAPY), 9days after the second booster immunization, which was one day beforeinfection with 2.3×10⁷ cfu/mouse of S. aureus strain Newman wildtype forinduction of sepsis. Data are presented as medians (center lines),interquartile ranges (boxes), and 80% central ranges (whiskers).N_(BSA)=13-15, N_(rClfA)=15, and N_(rClfAPY)=15.

FIG. 14 shows the specific antibody responses to recombinantClfAPY40-559 in serum samples of mice immunized with bovine serumalbumin (BSA), recombinant ClfA40-559 (rClfA), or recombinantClfAPY40-559 (rClfAPY), 9 days after the second booster immunization,which was one day before infection with 4.0×10⁶ cfu/mouse of S. aureusstrain Newman wildtype for induction of septic arthritis. Data arepresented as medians (center lines), interquartile ranges (boxes), and80% central ranges (whiskers). N_(BSA)=14-15, N_(rClfA)=15, andN_(rClfAPY)=15.

FIG. 15 shows the specific antibody responses to recombinant ClfA40-559in serum samples of mice immunized with bovine serum albumin (BSA),recombinant ClfA40-559 (rClfA), or recombinant ClfAPY40-559 (rClfAPY), 9days after the second booster immunization, which was one day beforeinfection with 4.0×10⁶ cfu/mouse of S. aureus strain Newman wildtype forinduction of septic arthritis. Data are presented as medians (centerlines), interquartile ranges (boxes), and 80% central ranges (whiskers).N_(BSA)=14-15, N_(rClfA)=15, and N_(rClfAPY)=15.

FIG. 16 of Example 2 shows the specific antibody responses torecombinant ClfAPY221-559 in serum samples of mice immunized with bovineserum albumin (BSA), recombinant ClfA221-559 (rClfA221-559), orrecombinant ClfAPY221-559 (rClfAPY221-559), 9 days after the secondbooster immunization. Data are presented as medians (center lines),interquartile ranges (boxes), and 80% central ranges (whiskers).N_(BSA)=15, N_(rClfA221-559)=14-15, and N_(rClfAPY221-559)=14-15.

FIG. 17 of Example 2 shows the specific antibody responses torecombinant ClfA221-559 in serum samples of mice immunized with bovineserum albumin (BSA), recombinant ClfA221-559 (rClfA221-559), orrecombinant ClfAPY221-559 (rClfAPY221-559), 9 days after the secondbooster immunization. Data are presented as medians (center lines),interquartile ranges (boxes), and 80% central ranges (whiskers).N_(BSA)=15, N_(rClfA221-539)=14-15, and N_(rClfAPY221-559)=14-15.

FIG. 18 of Example 3 shows the specific antibody responses torecombinant ClfA221-531 in serum samples of mice immunized withrecombinant ClfAPY221-531 (rClfAPY221-531), 9 days after the secondbooster immunization. Data are presented as medians (center lines),interquartile ranges (boxes), and 80% central ranges (whiskers).N_(rClfA221-531)=14-15.

EXAMPLES Example 1

rClfA a Region Truncates Comprising N1, N2 and N3 (Amino Acids 40-559)

Material and Methods

Full details of the numeric references in brackets given in the Examplesare provided at the end of this section.

Mice

NMRI mice were obtained from Scanbur BK (Sollentuna, Sweden) and weremaintained in the animal facility of the Department of Rheumatology,University of Göteborg, Sweden. Göteborg animal experiment ethical boardapproved the experiments. They were housed up to 10 animals per cagewith a 12 h light-dark cycle, and were fed standard laboratory chow andwater ad libitum. The animals were 6 to 16 weeks old at the start of theexperiments.

Bacterial Strains

For infection of animals the S. aureus wildtype strains Newman (14) andLS-1 (11) and constructed derivatives thereof were used. The clfAP₃₃₆SY₃₃₈A (clfAPYI) and clfA P₃₃₆AY₃₃₈S (clfAPYII) derivatives wereconstructed in strain Newman and transduced to strain LS-1 (see below).The deletion mutants Newman clfA2::Tn917 mutant DU5876 (3) and LS-1clfA2::Tn917 mutant (J. R. Fitzgerald et al., unpublished) were alsoused. Bacteria were grown on blood agar plates for 48 h, harvested, andkept frozen at −20° C. in PBS containing 5% (wt/vol) BSA (SigmaChemicals) and 10% (vol/vol) dimethyl sulfoxide. Before injection intoanimals, the bacterial suspensions were thawed, washed in PBS, andadjusted to appropriate cell concentrations. The number of viablebacteria was measured in conjunction with each challenge by cultivationon blood agar plates and counting colonies.

Construction of clfAPYI and clfAPYII Mutations in S. aureus Newman andLS-1

In this experiment, a full length ClfA A region truncate, comprising N1,N2 and N3, corresponding to amino acids 40 to 559, was used. In thefollowing description and figures:

-   -   ClfA may also be referred to as rClfA 40-559 (SEQ ID NO 3);    -   ClfA P₃₃₆SY₃₃₈A may also be referred to as clfAPYI, rclfAPY or        rclfAPYI (i.e clfAPYI 40-559) (SEQ ID NO 4); and    -   ClfA P₃₃₆AY₃₃₈S may also be referred to as clfAPYII, rclfAPYII        (i.e. clfAPYII 40-559) (SEQ ID NO 5).

A 1.02 kb PstI-BamHI fragment of pCF77 PY (Loughman et al., 2005)containing the mutations P₃₃₆S and Y₃₃₈A in clfA was cloned intopBluescriptII SK- (Stratagene). This plasmid was linearised with HindIIIand ligated to HindIII-cut pTSermC (J. Higgins, unpublished) to generateplasmid pARM, which is a temperature sensitive E. coli-S. aureus shuttlevector containing the P₃₃₆S and Y₃₃₈A substitutions.

In order to reduce the risk of unknowingly generating a functional orimmunoreactive epitope by substituting P₃₃₆ and Y₃₃₈, we generated asecond mutant, in which the order of the substitutions was reversed,yielding P₃₃₆A and Y₃₃₈S. To generate this a plasmid pJH2, analogous topARM but containing the P₃₃₆A and Y₃₃₈S substitutions, was generated.Overlap primer PCR was used with the same flanking primers used to makepCF77 PY (6), and a different pair of overlapping mutagenic primers:

F3:  GCAACTTTGACCATGGCCGCTTCTATTGACCCTGAAAATG and R3: CATTTTCAGGGTCAATAGAAGCGGCCATGGTCAAAGTTGC(mutations in bold and underlined) to generate pCF77 PYII. The 1.02 kbPstI-HindIII fragment of this plasmid was used as described above togenerate pJH2, a temperature sensitive E. coli-S. aureus shuttle vectorcontaining the P₃₃₆A and Y₃₃₈S substitutions.

Both pARM and pJH2 were transferred to RN4220 (15) by electroporationand subsequently transduced using phage 85 (16) to S. aureus Newman (14)and LS-1 (11). In these strains the plasmids were induced to insert intothe chromosome and then excise, leaving the mutations in the chromosomeof a proportion of transformants, generating Newman clfAPYI, NewmanclfAPYII, LS-1 clfAPYI and LS-1 clfAPYII. Transformants were screenedfor loss of the plasmid and a loss of fibrinogen-binding activity.Integrity of the clfA gene was verified by Southern hybridisation usinga clfA probe (data not shown). Expression of an immunoreactive protein(ClfAPY) was verified by Western immunoblotting using anti-ClfA region Apolyclonal rabbit antiserum (data not shown). The mutations wereverified by PCR across the KpnI-BamHI fragments from genomic DNA andcommercial sequencing of the products. The about 700 bases of the clfAgene of strain LS-1 that were sequenced were identical to thecorresponding bases in the Newman clfA gene of strain Newman.

Production of Recombinant ClfA and ClfAPY

His-tagged recombinant ClfA region A, domains N123 (amino acids 40-559),was produced from pCF40 as previously described (17), with an additionalpolishing step through an anion-exchange column. Plasmid pCF77 PY (6)was used as template to clone clfAPYI domains N123 into pQE30 togenerate pCF40PY. Using this plasmid, recombinant ClfAPY was alsoproduced by nickel affinity chromatography and anion exchangechromatograpy, as was described for rClfA. Eluates were dialysed againsttwo changes of PBS before concentration and freeze-drying.

Septic Arthritis and Sepsis Experiments

In experiments 1-3 all the mice (n=10 per group) were infected withstrain Newman to trigger arthritis. In experiments 4 and 5, the micewere infected with strain Newman and LS-1, respectively, to inducesepsis (n=10 per group).

Experiment 1 Mice were infected by intravenous injection with 3.5×10⁶cfu/mouse of S. aureus strain Newman or with 4.3×10⁶ cfu/mouse of NewmanclfAPYI mutant, both in 200 μl PBS. Clinical arthritis and weight changewas followed until day 7. Mice were sacrificed day 8, kidney growth ofbacteria were assessed and serum IL-6 and total IgG levels weremeasured. Synovitis and bone destruction was studied histologically onthe joints of fore and hind legs.

Experiment 2 Mice were infected with 5.0×10⁶ cfu, 6.0×10⁶ cfu or 4.3×10⁶cfu of S. aureus strain Newman, clfAPYI mutant or Newman clfA::Erm^(R)(clfA null mutant), respectively. Clinical arthritis and weight changewas followed until day 7. Mice were sacrificed day 7, kidney growth ofbacteria were assessed and serum IL-6 and total IgG levels weremeasured. Synovitis and bone destruction was studied histologically onthe joints of fore and hind legs.

Experiment 3 Mice were infected with 4.7×10⁶ cfu, 3.2×10⁶ cfu, 3.9×10⁶cfu or 4.8×10⁶ cfu of S. aureus strain Newman, clfAPYI mutant, NewmanclfAPYII mutant or Newman clfA null mutant, respectively. Clinicalarthritis and weight change was followed until day 7. Mice weresacrificed day 8 and kidney growth of bacteria were assessed.

The outcome of the experiments 1-3 were very similar, so data werepooled and presented together.

In Experiment 4 mice were injected intravenously with 5.2×10⁷ cfu,5.1×10⁷ cfu or 3.3×10⁷ cfu of S. aureus strain Newman, clfAPYI mutant orclfA null mutant, respectively. Mortality, weight change and clinicalarthritis were followed until day 10.

In Experiment 5 mice were infected with 9.4×10⁶ cfu, 7.9×10⁶ cfu,10.7×10⁶ cfu or 9.8×10⁶ cfu of S. aureus strain LS-1, LS-1 clfAPYImutant, LS-1 clfAPYII mutant, or LS-1 clfA null mutant, respectively.Mortality, clinical arthritis and weight change was followed until day16.

Intra-Articular Injection of Bacteria

One knee joint per mouse was injected with 2.4×10⁴ cfu, 2.4×10⁴ cfu, or3.4×10⁴ cfu of strain Newman wildtype, clfAPYI mutant or clfA knockoutmutant, respectively, in 20 μl PBS. N=10 per group. Mice were sacrificed3 days later, and the knee joints were collected for histopathologicalexamination.

Vaccination with Wild-Type and Mutant Recombinant ClfA

Purified rClfA40-559, rClfAPY40-559 (i.e. rClfAPYI) or BSA weredissolved in physiologic saline and emulsified 1:1 in Freund's completeadjuvant (Difco Laboratories). Two hundred μl of the emulsion containing30 μg (=0.53 nmol) of protein was injected subcutaneously (s.c.) on day0. First booster immunization with 30 μg of protein in physiologicsaline in incomplete Freund's adjuvant was performed on day 11. Secondbooster immunization was done day 21. On day 30 the mice were bled andsera were frozen for later analysis of antibody responses.

On day 31, 14-15 mice per group were infected by i.v. injection of4.0×10⁶ cfu/mouse for induction of septic arthritis, or by 2.3×10⁷cfu/mouse for induction of sepsis. Clinical arthritis, weight change andmortality were followed for 11 and 15 days, respectively. Bacterialgrowth in kidneys was assessed in the septic arthritis experiment.

Clinical Evaluation of Infected Mice

The clinical evaluation was performed in a blinded manner. Each limb wasinspected visually. The inspection yielded a score of 0 to 3 (0, noswelling and erythema; 1, mild swelling and/or erythema; 2, moderateswelling and/or erythema; 3 marked swelling and/or erythema). Thearthritic index was constructed by adding the scores from all four limbsof an animal. The overall condition of each mouse was also examined byassessing signs of systemic inflammation, i.e., weight decrease, reducedalertness, and ruffled coat. In cases of severe systemic infection, whena mouse was judged too ill to survive another 24 h, it was killed bycervical dislocation and considered dead due to sepsis.

Histological Examination

Histological examination of joints was performed using a modification(8) of a previously described method (18).

Bacteriologic Examination of Infected Kidneys

Kidneys were aseptically dissected, kept on ice, homogenised, seriallydiluted in PBS and spread on blood agar plates. After 24 h of incubationin 37° C. the number of cfu per kidney pair was determined.

Measurement of Serum IgG

Levels in serum of total IgG were measured by the radial immunodiffusiontechnique (19). Goat-Anti-Mouse-IgG and mouse IgG standard werepurchased from Southern Biotech, Birmingham, Ala.

Specific Antibodies—ELISA

Serum samples from immunized mice were obtained 9 days after the secondbooster immunization. The serum specific antibody response against rClfAand rClfAPY was measured by ELISA. Microplates (96-well; Nunc) werecoated with 5 μg/ml of recombinant protein in PBS. Blocking agent, serumsamples, biotinylated antibodies, and ExtrAvidin-proxidase were alldiluted in PBS. The assay was run according to a previous description(8). All serum samples were diluted 1:20000, and antibody response wasmonitored as absorbance at 405 nm.

In a second run, to get a more accurate measure of the specific antibodyresponses in the different immunization groups, the responses weredetermined at several serum dilutions. Thus, all serum samples werediluted 1:5000, 1:20000, 1:80000 and 1:320000, and antibody response wasmonitored as absorbance at 405 nm.

IL-6 Analysis

Serum IL-6 was detected by a method previously described (20).

Statistical Analysis Statistical evaluation was done by using theMann-Whitney U test. P<0.05 was considered to be significant. Data arereported as medians, interquartile ranges, and 80% central ranges,unless otherwise mentioned.

Results

Exchange of two amino acids necessary for ClfA binding to fibrinogenhampers development of septic arthritis and sepsis

Two amino acids (P336 and Y338) that are known to be required forfibrinogen binding by ClfA were altered by allelic exchange to createmutants of strains Newman and LS1 that expressed anon-fibrinogen-binding ClfA protein on the cell surface. The level ofexpression and integrity of the protein was measured by Western blottingwhich established that there was good expression of the mutant proteinson the bacterial surface and expressed protein was the right size.

The ability of Newman wild-type and Newman clfA P₃₃₆S Y₃₃₈A (clfAPYI) toprovoke septic arthritis was investigated. Septic arthritis was inducedby intravenous inoculation of 3.5×10⁶ to 5.0×10⁶ colony-forming units(cfu) and 3.2×10⁶ to 6.0×10⁶ cfu of Newman wild-type and the clfAPYImutant, respectively. The development of arthritis was studiedclinically for 7 days. The clfAPYI mutant provoked significantly lesssevere arthritis than the wild-type strain over the entire experimentalperiod (P>0.001, FIG. 1 A). The frequency of arthritis was lower forNewman clfAPYI at most time points (FIG. 6).

Unexpectedly, it appears that the new amino acid composition in theClfAPYI molecule fits for interaction with a host anti-bacterialdefence. To check for this possibility, a new construct was made wheredifferent amino acids were substituted for P336 and Y338 (clfA P₃₃₆AY₃₃₈S: clfAPYII). Mice that were inoculated with 3.9×10⁶ cfu of NewmanclfAPYII developed arthritis to the same low extent as the clfAPYImutant (FIG. 1 A), and with a similar frequency (FIG. 6). This outcomesuggests strongly that the loss of fibrinogen binding is responsible forthe reduced level of arthritis.

It is possible that ClfA is involved in the development of arthritis bymechanisms that do not involve fibrinogen binding. To test this a ClfAdeletion mutant lacking the ClfA protein was compared to mutantsexpressing the modified non-fibrinogen binding ClfA protein. However,mice that were infected with 4.3×10⁶ to 4.8×10⁶ cfu of clfA null mutantdeveloped arthritis in a manner not different from the clfAPYI andclfAPYII mutant infected mice (FIG. 1 A). The frequency of arthritis wasalso indistinguishable (FIG. 6).

Infected joints were also investigated histologically. The synovitis inNewman clfAPYI-infected mice was significantly milder than in wild-typeinfected mice in both experiment 1 and 2 (P=0.02 and 0.001,respectively). Bone destruction, a major cause of sequels in humanseptic arthritis, was almost absent in the Newman clfAPYI-infectedsamples (Experiment 2, P=0.001). The synovitis and bone destructioninduced by the Newman clfA null mutant were also less pronouncedcompared to mice infected with Newman wild-type (P=0.003 and 0.006,respectively), but somewhat more severe than in the Newman clfAPYIgroup, although not significantly so.

Next, the metabolic consequences of the clfA mutations for theinfectious process were analysed. Mice infected with the Newmanwild-type strain lost up to about 30% of their body weight during theexperimental period. Mice that were infected with the fibrinogenbinding-deficient mutants Newman clfAPYI and Newman clfAPYII lost hardlyany weight at all (P>0.0001 versus wild-type). In contrast, the NewmanclfA null mutant had an intermediate effect on the weight loss, causingsignificantly less than the wild-type strain, but significantly morethan the clfAPYI and clfAPYII mutant strains (P≤0.02 in most cases, FIG.1 B).

The serum levels of IL-6, a measure of systemic inflammatory response,were analyzed at day 7-8 of infection. The pattern of IL-6 expressionwas similar to weight changes. Newman wild-type evoked high levels ofserum IL-6 (4.8 (2.8, 5.7) ng/ml), the Newman clfAPYI mutant evokedconsiderably lower IL-6 (0.2 (0.07, 2.4) ng/ml, P<0.0001) while theNewman clfA null mutant gave rise to an intermediate response (2.5 (1.3,3.2) ng/ml) with significant differences to both the wild-type andclfAPYI mutant group (P=0.009 and P=0.008, respectively) (median,interquartile range).

The growth of bacteria in kidneys was significantly greater in Newmanwild-type-infected mice, compared to both of the Newman clfAPY mutantsand the Newman clfA null mutant (P<0.0001, P=0.011, and P=0.005,respectively; FIG. 2). The Newman clfA null mutant-infected mice hadsignificantly more bacterial growth in kidneys than NewmanclfAPYI-infected mice (P=0.0005, FIG. 2).

Total IgG in sera was measured in mice on day 7-8 of infection. Therewas a significantly lower increase of IgG levels in both the NewmanclfAPYI- and Newman clfA null mutant-infected groups as compared to miceinfected with the wild-type strain (3.1 (1.2, 4.9); 2.3 (1.0, 2.6); and6.4 (5.0, 11.0), respectively (median, interquartile range); P≤0.0003).There were no significant differences between the two mutant groups.

The mortality was 17% in the Newman wild type-infected mice, 0% in theNewman clfAPYI and clfAPYII mutant groups and 30% in the Newman clfAnull mutant group. There were significant differences in mortalitybetween the wild-type and the clfAPYI groups, and between the clfAPYIand clfA null mutant groups (P<0.05 and P<0.01, respectively).

It appears that direct and indirect signs of systemic inflammation arelower in mice infected with S. aureus expressing ClfA that is deficientin fibrinogen binding. Unexpectedly, the strain which lacked ClfAexpression altogether induced more systemic inflammation than a ClfAPYmutant-expressing strain.

Sepsis was induced in mice by increasing the inoculation dose of S.aureus. Mice were infected with 5.2×10⁷ cfu of Newman wild type, 5.1×10⁷cfu of the Newman clfAPYI mutant and 3.3×10⁷ cfu of the Newman clfA nullmutant. Within 5 days all wild-type infected mice were dead, but onlyone clfAPYI mutant mouse out of ten were dead after 10 days of infection(P<0.0001, FIG. 3). Mice infected with the clfA null mutant alsosurvived a significantly shorter time than the clfAPYI mutant-infectedmice (P<0.0001, FIG. 3). In this experiment the mice challenged with theclfA null mutant developed significantly more arthritis than the clfAPYImutant group, while at the same time they lost significantly more weight(FIGS. 7 and 8). Thus, by analogy with the measures of systemicinflammation in the septic arthritis experiments, the survival of themice is prolonged if the ClfA molecule is expressed, as long as it lacksfibrinogen binding properties.

Injection of Bacteria into Joints

To test if the inflammatory reaction in the joint is dependent onfibrinogen binding, Newman wild-type, Newman clfAPYI or Newman clfA nullwere injected directly into a knee joint of mice, thereby by-passing thesystemic compartment. Synovitis, including polymorphonuclearinfiltration of the joint cavity, and bone destruction was studied byhistology 3 days later. The mice received 2.4×10⁴ cfu of wild-type,2.4×10⁴ cfu of the clfA null mutant, or 3.4×10⁴ cfu of clfAPYI mutant inone knee. The synovitis and the polymorphonuclear infiltrationhistologic index in the joint cavity was 0.25 (0, 3.0) for kneesinfected with wild-type, 2.38 (0.25, 3.0) for the clfA null mutant and0.25 (0, 0.25) for the clfAPYI mutant (median, interquartile range). Thehistologic index for destruction of bone was 0 (0, 1.0) for wild-type,1.0 (0, 1.0) for the clfA null mutant, and 0 (0, 0) for the clfAPYImutant (median, interquartile range; P=0.01 between the clfAPYI mutantand the clfA null mutant). Since the clfAPYI mutant evoked very littlesynovitis and destruction, despite the fact that 42% more of that strainwas given to mice than the other strains, it is concluded thatClfA-promoted fibrinogen binding is needed for the maximal inflammatoryresponse within the joint. Again, the absence of ClfA expressionenhanced inflammation compared to the fibrinogen binding deficient ClfAmutant.

PY Mutation in Strain LS-1

To determine if the ability of ClfA to bind fibrinogen affects virulenceof other strains of S. aureus, the clfAPYI, clfAPYII and clfA nullmutations were transduced to the TSST-1 expressing S. aureus strainLS-1. Mice were challenged with 9.4×10⁶ cfu of LS-1 wild-type, 7.9×10⁶cfu of LS-1 clfAPYI, 10.7×10⁶ cfu of LS-1 clfAPYII, or 9.4×10⁶ cfu ofthe LS-1 clfA null mutant. Sepsis was studied by following the survivalrate. After 16 days only 40% of mice challenged with the wild-typestrain were alive while 90% of the mice challenged with the clfAPYImutant and clfA null mutant groups and 80% mice infected with theclfAPYII mutant were alive (FIG. 4). The clfAPYI mutants and the clfAnull mutant of LS-1 were significantly less virulent (P=0.014, P=0.05and P=0.03, respectively).

Immunization with Recombinant ClfA Proteins

The effect of vaccination with recombinant wild-type ClfA A domainprotein (rClfA) and mutant ClfAPYI protein (rClfAPY) was studied in boththe septic arthritis model and the sepsis model. Mice were sensitizedand then boosted twice with control protein BSA, rClfA, or rClfAPY, andsubsequently infected with 4.0×10⁶ cfu of S. aureus strain Newman toinduce septic arthritis, or with 2.3×10⁷ cfu of strain Newman to inducesepsis. Immunization with rClfAPY (i.e. ClfAPYI recombinant protein Adomain) protected significantly against septic death as compared tocontrol mice (P=0.01, FIG. 5) while rClfA immunization did not achievesignificant protection. One day before bacterial infection there was amuch higher specific serum antibody response to both rClfAPY and rClfAin mice immunized with rClfAPY (A₄₀₅=0.39 (0.33, 0.56) and 0.71 (0.52,0.81)) as compared to mice immunized with rClfA (A₄₀₅=0.13 (0.07, 0.17)and 0.15 (0.10, 0.24), P<0.0001 in both comparisons (median,interquartile range)). Control immunized animals had only backgroundlevels (A_(405 nm)=0 and 0.01 (0, 0.01) (median, interquartile range)).The immunized mice which were to be infected with the lower, arthriticbacterial dose had similar antibody responses to rClfA and rClfAPY asthe mice in which sepsis were induced (data not shown). Immunizationwith both rClfA and rClfAPY protected against the development ofarthritis, although the protection was not significant (FIG. 9).

During day 5 to 9 after infection the weight loss was significantlyreduced in the rClfAPY and rClfA immunized mice, as compared to thecontrol mice (data not shown).

A trend to diminished bacterial growth in kidneys of mice immunized withrClfAPY or rClfA at day 11 after infection (BSA: 38 (3, 436); rClfAPY: 7(2, 17); rClfA: 10 (7, 54)×10⁷ cfu/kidney pair) was observed.

To get a more accurate measure of the specific antibody responses in thedifferent immunization groups, the responses were determined at severalserum dilutions (the second run). Data shows that there were very likelyhigher titers of specific antibodies in sera from rClfAPY immunized miceto both the rClfAPY and rClfA wildtype antigens, in both the mice whichwere to be infected with the septic and the arthritic bacterial dose,respectively, than in sera from rClfA wildtype immunized mice, sincethere was significantly higher antibody responses measured as absorbancein mice immunized with rClfAPY at each serum dilution in all comparisons(P<0.0001 to P=0.008, FIG. 12-15). BSA immunization evoked only abackground antibody response.

Conclusion

The results strongly suggest that the ClfA-fibrinogen interaction iscrucial for the bacterial virulence and disease outcome. The ability ofClfA to bind fibrinogen was associated with enhanced virulence in termsof the ability to cause septic death. In both staphylococcal strainstested, a clfAPY mutant induced less septic death than the wild-type.Also, the severity of arthritis was strongly reduced in mice infectedwith the non-fibrinogen binding clfAPY mutant.

A likely mechanism for the promotion of virulence by thefibrinogen-bacterial cell surface interaction is inhibition ofneutrophil phagocytosis (5). Neutrophils are crucial for the hostdefence in the early phase of S. aureus infection (13). Withoutneutrophils, bacterial growth is strongly augmented in blood andkidneys, and the frequency of arthritis and mortality increases.Fibrinogen mediated inhibition of neutrophil phagocytosis by ClfA couldexplain at least in part the more pronounced virulence of wildtype S.aureus compared to the clfAPY mutants. Binding of fibrinogen to ClfAcould decrease opsonophagocytosis by neutrophils by reducing opsonindeposition or access to opsonins by neutrophil receptors. Alternativelybound fibrinogen might block the binding of an unknown protective hostfactor to S. aureus. Another option is that the fibrinogen-ClfAinteraction promotes bacterial passage from blood vessel into the tissueor promotes colonization in tissues.

Unexpectedly, our data also show the ClfA null mutant was more virulentthan the clfAPY mutant strains. Possibly the ClfA protein has functionsin vivo other than interacting with fibrinogen. This interaction isclearly disadvantageous for the host as shown in this study. Otherfunctions of ClfA are presently not well mapped but non-fibrinogendependent platelet aggregation exerted by ClfA might result in trappingof big amounts of S. aureus in circulation with subsequent eliminationof the bacterial-platelet complexes through the reticuloendothelialsystem. Such platelet aggregation mediated elimination of staphylococciwould readily occur in the wild-type and clfAPY mutated strains but notin the clfA knockout. Whereas in the wild-type strain the fibrinogeninteraction would overshadow the other events, in the clfAPY mutantssuch bacterial elimination might be highly beneficial to the host.

The clfA knockout mutant protected against septic death to the samedegree as the clfAPY mutation in S. aureus strain LS-1, but protectedless, if at all, in strain Newman. The overall impact of ClfA expressionon bacterial virulence could differ between different S. aureus strainsdepending on the level of expression and the presence of other virulencefactors.

The issue whether the clfAPY mutant displays equal or lower virulenceonce in the joint cavity is of certain importance having in mind that ininflamed synovial fluid fibrinogen and fibrin are abundant. Our datasuggest that the clfAPY mutant is less destructive for cartilage andbone.

The protective effect of recombinant ClfA A domain non-fibrinogenbinding P₃₃₆Y₃₃₈ mutant was greater than for wildtype rClfA.Immunization with ClfAPY very likely induced a better immune responsesince higher specific antibody responses were evoked against both theimmunogen and the wildtype ClfA protein. More importantly, it induced agreater protective immune response against septic death than wildtypeClfA.

In conclusion, our results show that rClfAPY is a better vaccinecandidate than wild type recombinant ClfA. We hypothesize that bindingof fibrinogen by wild-type ClfA protein during the immunization phasedecreases antigen presentation due to hiding of important epitopes onthe ClfA molecule and hence impairs specific antibody production.

Example 2

rClfA a Region Truncate Comprising N2 and N3 (rClfA 221-559)

Materials & Methods:

The protocols outlined in Example 1 were followed in this example whichutilized

-   -   rClfA 221-559 (i.e. ClfA A region truncate comprising N2 and N3        corresponding to amino acids 220-559)    -   rClfAPY221-559; and    -   BSA.

There were 15 female NMRI mice per group who were 8 weeks old at startof experiments. In this Example, the constructs used for immunizationwere ClfA wild type/native N2N3 truncate, ClfA N2N3 truncate withmutation PY as defined in Example 1. BSA was used as the control.

Vaccination with Wild-Type and Mutant Recombinant ClfA

The mice were immunized with rClfA 221-559, rClfAPY 221-559 or BSA inaccordance with the protocol of Example 1.

Purified rClfA221-559, rClfAPY221-559 (i.e. ClfAPYI recombinant proteinA subdomains N2 and N3) or BSA were dissolved in PBS and emulsified 1:1in Freund's complete adjuvant. Two hundred μl of the emulsion containing30 μg (=0.79 nmol) of protein was injected s.c. on day 0. First boosterimmunization with 30 μg of protein in physiologic saline in incompleteFreund's adjuvant was performed on day 12. Second booster immunizationwas done day 22. On day 31 the mice were bled and sera were frozen forlater analysis of antibody responses.

Specific Antibodies—ELISA

Serum samples from immunized mice were obtained 9 days after the secondbooster immunization. The serum specific antibody response againstrClfA221-559 and rClfAPY221-559 was measured by ELISA. Microplates(96-well; Nunc) were coated with 5 μg/ml of recombinant protein in PBS.Blocking agent, serum samples, biotinylated antibodies, andExtrAvidin-proxidase were all diluted in PBS. The assay was runaccording to a previous description (8). All serum samples were diluted1:5000, 1:20000, 1:80000 and 1:320000, and antibody response wasmonitored as absorbance at 405 nm.

Results:

Specific Antibody Response:

The antibody response was measured by absorbance in an ELISA-assay, asper Example 1, with four different serum dilutions. The data obtainedwas very similar to the data in the Example 1.

It was found that rClfAPY221-559 immunization very likely gave rise tohigher titers of specific antibodies to both native rClfA221-559 andrClfAPY221-559, as compared to native rClfA221-599 immunization, sincethere were significantly higher antibody responses measured asabsorbance in mice immunized with rClfAPY221-559 at each serum dilutionin all comparisons but one (P=0.001 to 0.025, see FIGS. 16 and 17). BSAimmunization evoked only background levels of antibody response.

Conclusion

We found that immunization with a rClfAPY221-559 protein gave rise tosignificantly higher antibody responses to both the immunogen and thewildtype ClfA protein, than immunization with the native protein.

Based on these findings, we conclude that PY-immunization, regardless ifthe PY protein comprises amino acids 40 to 550 as in Example 1 or aminoacids 221 to 559 as in Example 2, induces a better immune response thanimmunization with native ClfA of the corresponding size.

Example 3

ClfA a Region Truncate (6/Delta Latch Truncate)

Materials & Methods:

The protocols outlined in Example 1 were followed in this example whichutilized the following construct:

-   -   rClfA 221-531 (i.e. rClfA A region truncate comprising N2 and N3        amino acids 220-559 but without the latching peptide amino acids        532-538 and the subsequent proline-rich residues.

There were 15 female NMRI mice in the group who were 8 weeks old atstart of experiment. In this Example, the above construct was used forimmunization. The mice were immunized with the above truncate inaccordance with the protocol of Example 1.

Vaccination with Wild-Type and Mutant Recombinant ClfA

Purified rClfA221-531 was dissolved in PBS and emulsified 1:1 inFreund's complete adjuvant. Two hundred μl of the emulsion containing0.79 nmol of protein was injected s.c. on day 0. First boosterimmunization with 0.79 nmol of protein in physiologic saline inincomplete Freund's adjuvant was performed on day 12. Second boosterimmunization was done day 22. On day 31 the mice were bled and sera werefrozen for later analysis of antibody responses.

Specific Antibodies—ELISA

Serum samples from immunized mice were obtained 9 days after the secondbooster immunization. The serum levels of specific antibodies wasmeasured by ELISA. Microplates (96-well; Nunc) were coated with 4.6μg/ml of rClfA221-531 protein which is equimolar to 5 μg/ml ofrClfA221-559 and rClfAPY221-559 from Examples 1 and 2. Blocking agent,serum samples, biotinylated antibodies, and ExtrAvidin-proxidase wereall diluted in PBS. The assay was run according to a previousdescription (8). All serum samples were diluted 1:5000, 1:20000, 1:80000and 1:320000, and antibody response was monitored as absorbance at 405nm.

Results:

The antibody response was measured by absorbance in an ELISA-assay, asper Example 1. It was found that rClfA221-531 immunization gave rise toan immune response, measured as a specific antibody response (FIG. 18).

Conclusion:

We found that rClfA221-531 works as an immunogen, since the antigenevokes a specific antibody response.

REFERENCES

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The invention claimed is:
 1. A vaccine composition comprising animmunologically effective amount of a recombinant fibrinogenbinding-deficient mutant of staphylococcal clumping factor A (ClfA) or afragment thereof dispersed or emulsified in a saline solution and/orpharmaceutically acceptable adjuvant for injection, said fragmentcomprising at least amino acid residues 221 to 531 of the fibrinogenbinding region, wherein the recombinant fibrinogen binding-deficientmutant of staphylococcal ClfA or fragment thereof has at least one aminoacid residue substitution or deletion at amino acid residue Ala254,Tyr256, Pro336, Tyr338, Ile387, Lys389, Glu526 and/or Val527, saidrecombinant fibrinogen binding-deficient mutant of staphylococcal ClfAor fragment thereof having reduced ability or lacking the ability tonon-covalently bind fibrinogen and stimulating a greater antibody immuneresponse than a wild type ClfA protein.
 2. The vaccine of claim 1,wherein the recombinant fibrinogen binding-deficient mutant ofstaphylococcal ClfA has a sequence according to SEQ ID No. 1 or asequence with at least 85% sequence identity to the sequence of SEQ IDNo.
 1. 3. The vaccine of claim 1, wherein the recombinant fibrinogenbinding-deficient mutant of staphylococcal ClfA comprises the fibrinogenbinding region only or a fragment thereof.
 4. The vaccine of claim 1,wherein the recombinant fibrinogen binding-deficient mutant ofstaphylococcal ClfA is derived from S. aureus, S. epidermidis and/or S.lugdunensis.
 5. The vaccine of claim 1, wherein the recombinantfibrinogen binding-deficient mutant of staphylococcal ClfA comprises theamino acid sequence according to any of SEQ ID Nos. 4 to
 14. 6. Thevaccine of claim 1, wherein recombinant fibrinogen binding-deficientmutant of staphylococcal ClfA amino acid residues Ala254, Tyr256,Pro336, Tyr338, Ile387, Lys389, Glu526 and/or Val527 are substitutedwith either Ala or Ser.
 7. The vaccine of claim 1, wherein residue P336and/or Y338 of the fibrinogen binding region (Region A) of ClfA issubstituted with either serine or alanine to result in rClfAP336S Y338Aor rClfAP336 A Y338S.
 8. The vaccine of claim 1, wherein the recombinantfibrinogen binding-deficient mutant of staphylococcal ClfA has the aminoacid sequence according to any of SEQ ID Nos. 1 to 3 wherein residueP336 and/or Y338 are substituted with either serine and/or alanine, orfragment thereof.
 9. The vaccine of claim 1, wherein the recombinantfibrinogen binding-deficient mutant of staphylococcal ClfA comprises thefibrinogen binding protein, the fibrinogen binding region, the minimalfibrinogen binding region and/or a fragment thereof.
 10. The vaccine ofclaim 1, wherein the recombinant fibrinogen binding-deficient mutant ofstaphylococcal ClfA fragment comprises at least part of the fibrinogenbinding region to result in a recombinant fragment of the fibrinogenbinding protein with reduced ability or lacking the ability tonon-covalently bind fibrinogen.
 11. The vaccine of claim 1, wherein therecombinant fibrinogen binding-deficient mutant of staphylococcal ClfAcomprises a. Subregions N123, spanning amino acid residues 40 to 559 ofthe fibrinogen binding region (Region A); b. Subregions N23, spanningamino acid residues 221 to 559 of the fibrinogen binding region of ClfA(Region A); and/or c. Amino acid residues 221 to 531 of the fibrinogenbinding region (Region A).
 12. The vaccine of claim 1, wherein therecombinant fibrinogen binding-deficient mutant of staphylococcal ClfAcomprises an isolated recombinant staphylococcal clumping factor A(ClfA).
 13. The vaccine of claim 1, wherein said recombinant fibrinogenbinding-deficient mutant of staphylococcal ClfA or fragment thereof isdispersed in a sterile, isotonic saline solution.
 14. A method ofpreparing a vaccine composition comprising an immunologically effectiveamount of a recombinant fibrinogen binding-deficient mutant ofstaphylococcal clumping factor A (ClfA) or fragment thereof, said methodcomprising formulating the recombinant fibrinogen-deficient mutant ofstaphylococcal ClfA or fragment thereof as a pharmaceutical compositionfor injection, by dispersing or emulsifying an immunologically effectiveamount of the recombinant fibrinogen-deficient mutant of staphylococcalClfA or fragment thereof in a saline solution and/or pharmaceuticallyacceptable adjuvant, wherein said fragment comprises at least amino acidresidues 221 to 531 of the fibrinogen binding region, wherein therecombinant fibrinogen binding-deficient mutant of staphylococcal ClfAor fragment thereof has at least one amino acid residue substitution ordeletion at amino acid residue Ala254, Tyr256, Pro336, Tyr338, Ile387,Lys389, Glu526 and/or Val527, and wherein the recombinant fibrinogenbinding-deficient mutant of staphylococcal ClfA or fragment thereof hasreduced ability or lacks the ability to non-covalently bind fibrinogen,and stimulates a greater antibody immune response than a wild type ClfAprotein.
 15. The method of claim 14, wherein the formulating comprisesdispersing the immunologically effective amount of the recombinantfibrinogen binding-deficient mutant of staphylococcal ClfA or fragmentthereof in a sterile, isotonic saline solution.